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Background <p>The world is facing both an increasingly severe energy crisis and a growing problem of agricultural pollution. The utilization of agricultural waste by anaerobic digestion (AD), has received increasing attention. AD using representative waste cow dung results in total ammonia nitrogen (TAN) accumulation and inhibition of methanogens resulting in reduced CH<sub>4</sub> production. However, there is a lack of highly efficient in-situ biological domestication strategies to enhance the TAN tolerance of methanogens in AD systems.</p> Results <p>In this study, an incremental approach to gradually increasing the TAN concentration has been used for overcoming the problem. The results showed that at an ultra-high concentration of 6124.09&#xa0;mg/L TAN, a 48-day domesticated AD system functioned stably and the cumulative CH<sub>4</sub> production reached 72.81&#xa0;mL/g volatile solids, whereas the undomesticated AD system failed to produce CH<sub>4</sub>. After domestication, the lactate dehydrogenase concentration decreased to 96.44&#xa0;ng/L and the adenosine triphosphate concentration increased to 48.77&#xa0;nmol/L, confirming that microbial activity improved. Hydrolytic and acidogenic bacteria were enriched, with <i>Methanosarcina</i> (79.73%) dominating the domesticated AD system, primarily <i>Methanosarcina mazei</i>. Metagenomic analysis showed that with two-component system enrichment, the key inhibited steps from glycerate-1,3P2 to pyruvate (2.498‰), and from acetyl-CoA and acetyl phosphate to acetic acid (1.141‰ and 0.798‰), as well as vital methanogenic genes <i>mcrA</i> (0.128‰), <i>mcrB</i> (0.127‰), and <i>mcrG</i> (0.065‰), were both enriched, which favored a stable methanogenic system. More importantly, this ultra-high resistance AD system also showed the potential to increase the CH<sub>4</sub> production per unit substrate at the Minhe biogas plant with 24,000&#xa0;m<sup>3</sup> operation scale in Shandong Province, China.</p> Conclusions <p>Stepwise increase the TAN concentration is a novel method that was demonstrated to be a practical and sustainable way to overcome ultra-high TAN inhibition (6124.09&#xa0;mg/L). During domestication, the two-component system may regulate the microbial collaborative network to ensure microbial activity and high abundance enrichment, thus potentially constructing a methanogenic system dominated by hydrogenotrophic and acetoclastic methanogenesis, holding a promising application prospect. This study helped recognize the potential of methanogens in tolerating ultra-high inhibition and developed an achievable AD technology for robustly treating fecal residue and wastewater in practice.</p>

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An innovative strategy for overcoming ultra-high ammonia nitrogen inhibition on anaerobic methanogenesis via stepwise domestication

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年10月30日

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  • Heng Wu,
  • Taili Dong,
  • Anjie Li,
  • Jing Chen,
  • Huaiwen Zhang,
  • Hongyi Lv,
  • Caiyun Yang,
  • Xiaohui Guo,
  • Xuanmin Yang,
  • Ling Qiu,
  • Changlin Miao,
  • Yiqing Yao

摘要

Background

The world is facing both an increasingly severe energy crisis and a growing problem of agricultural pollution. The utilization of agricultural waste by anaerobic digestion (AD), has received increasing attention. AD using representative waste cow dung results in total ammonia nitrogen (TAN) accumulation and inhibition of methanogens resulting in reduced CH4 production. However, there is a lack of highly efficient in-situ biological domestication strategies to enhance the TAN tolerance of methanogens in AD systems.

Results

In this study, an incremental approach to gradually increasing the TAN concentration has been used for overcoming the problem. The results showed that at an ultra-high concentration of 6124.09 mg/L TAN, a 48-day domesticated AD system functioned stably and the cumulative CH4 production reached 72.81 mL/g volatile solids, whereas the undomesticated AD system failed to produce CH4. After domestication, the lactate dehydrogenase concentration decreased to 96.44 ng/L and the adenosine triphosphate concentration increased to 48.77 nmol/L, confirming that microbial activity improved. Hydrolytic and acidogenic bacteria were enriched, with Methanosarcina (79.73%) dominating the domesticated AD system, primarily Methanosarcina mazei. Metagenomic analysis showed that with two-component system enrichment, the key inhibited steps from glycerate-1,3P2 to pyruvate (2.498‰), and from acetyl-CoA and acetyl phosphate to acetic acid (1.141‰ and 0.798‰), as well as vital methanogenic genes mcrA (0.128‰), mcrB (0.127‰), and mcrG (0.065‰), were both enriched, which favored a stable methanogenic system. More importantly, this ultra-high resistance AD system also showed the potential to increase the CH4 production per unit substrate at the Minhe biogas plant with 24,000 m3 operation scale in Shandong Province, China.

Conclusions

Stepwise increase the TAN concentration is a novel method that was demonstrated to be a practical and sustainable way to overcome ultra-high TAN inhibition (6124.09 mg/L). During domestication, the two-component system may regulate the microbial collaborative network to ensure microbial activity and high abundance enrichment, thus potentially constructing a methanogenic system dominated by hydrogenotrophic and acetoclastic methanogenesis, holding a promising application prospect. This study helped recognize the potential of methanogens in tolerating ultra-high inhibition and developed an achievable AD technology for robustly treating fecal residue and wastewater in practice.